This application requests a Zeiss LSM 510 NLO two-photon microscope optimized for quantitative imaging with living cells and tissues. A Titanium-sapphire laser (Coherent Chameleon extended range) will be directly coupled to the input port of the scan head, attached to a Zeiss Axiovert 200 microscope having coverslip-corrected water immersion objectives. The device will be incorporated in a new Live Microscopy Core facility that will serve investigators from multiple departments throughout the University of Cincinnati College of Medicine, who need the instrument to perform live cell and live tissue fluorescence microscopy that is beyond the capabilities of confocal microscopy. Our 4 major user groups require multi-photon microscopy to (1) provide high-resolution imaging of cellular and subcellular events within tissue at focal depths greater than 50mu/m from the surface (at these depths, confocal microscopy can neither provide high spatial resolution nor avoid photodamage), and (2) permit localized deposition of energy for uncaging, photodamage, or photobleaching experiments so that events can then be tracked by conventional confocal microscopy on the same microscope. All major users have documented that multi-photon microscopy based on the Zeiss platform will provide a significant advance beyond confocal microscopy in addressing their experimental needs. The needs of our users can not be met by equipment at our institution, because there is no multi-photon microscope at the entire University, and confocal microscopy is inadequate to address the questions of the users. The multi-photon microscope would be installed in a location near the laboratory of the PI, which is central to all investigators, and would facilitate daily monitoring of equipment status by the PI. The application is supported by substantial interest, need, and financial support at the University, and will serve a strongly NIH-funded scientific community.
